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RESEARCH ARTICLE

Global gene expression profiling and antibiotic susceptibility after repeated exposure to the carbon monoxide-releasing molecule-2 (CORM-2) in multidrug-resistant ESBL-producing uropathogenic Escherichia coli Charlotte Sahlberg Bang*, Isak Demirel, Robert Kruse, Katarina Persson

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School of Medical Sciences, Faculty of Medicine and Health, iRiSC—Inflammatory Response and Infection ¨ rebro University, O ¨ rebro, Sweden Susceptibility Centre, O * [email protected]

Abstract OPEN ACCESS Citation: Sahlberg Bang C, Demirel I, Kruse R, Persson K (2017) Global gene expression profiling and antibiotic susceptibility after repeated exposure to the carbon monoxide-releasing molecule-2 (CORM-2) in multidrug-resistant ESBL-producing uropathogenic Escherichia coli. PLoS ONE 12(6): e0178541. https://doi.org/10.1371/journal. pone.0178541 Editor: Ligia M. Saraiva, Universidade Nova de Lisboa Instituto de Tecnologia Quimica e Biologica, PORTUGAL Received: October 27, 2016 Accepted: May 15, 2017 Published: June 7, 2017 Copyright: © 2017 Sahlberg Bang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Treatment of urinary tract infections is today a challenge due to the increasing prevalence of multidrug-resistant ESBL-producing uropathogenic Escherichia coli (UPEC). There is an urgent need for new treatment strategies for multidrug-resistant UPEC and preferably with targets that have low potential for development of resistance. Carbon monoxide-releasing molecules (CORMs) are novel and potent antibacterial agents. The present study examines the transcriptomic targets of CORM-2 in a multidrug-resistant ESBL-producing UPEC isolate in response to a single exposure to CORM-2 and after repeated exposure to CORM-2. The bacterial viability and minimal inhibitory concentration (MIC) were also examined after repeated exposure to CORM-2. Microarray analysis revealed that a wide range of processes were affected by CORM-2, including a general trend of down-regulation in energy metabolism and biosynthesis pathways and up-regulation of the SOS response and DNA repair. Several genes involved in virulence (ibpB), antibiotic resistance (marAB, mdtABC) and biofilm formation (bhsA, yfgF) were up-regulated, while some genes involved in virulence (kpsC, fepCEG, entABE), antibiotic resistance (evgA) and biofilm formation (artIP) were down-regulated. Repeated exposure to CORM-2 did not alter the gene expression patterns, the growth inhibitory response to CORM-2 or the MIC values for CORM-2, cefotaxime, ciprofloxacin and trimethoprim. This study identifies several enriched gene ontologies, modified pathways and single genes that are targeted by CORM-2 in a multidrug-resistant UPEC isolate. Repeated exposure to CORM-2 did not change the gene expression patterns or fold changes and the susceptibility to CORM-2 remained after repeated exposure.

Data Availability Statement: All microarray data files are available from the Gene Expression Omnibus database (accession number GSE87627). Funding: We gratefully acknowledge the support from the Faculty of Medicine and Health at O¨rebro University and Nyckelfonden at O¨rebro University Hospital. The funders had no role in study design,

PLOS ONE | https://doi.org/10.1371/journal.pone.0178541 June 7, 2017

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Repeated exposure to CORM-2 in multiresistant UPEC

data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Nearly one-fifth of all uropathogenic strains of E. coli (UPEC) are resistant to the most commonly used antibiotics [1]. Therapeutic options are limited for extended spectrum beta-lactamase (ESBL)-producing E. coli, where the bacteria have acquired a plasmid with genes that code for the enzyme ESBL. ESBL-producing Enterobacteriaceae spp. contain genes that code for the ESBL enzyme, and several different ESBL enzyme variants (TEM, SHV, CTX-M) have been identified. ESBL-producing E. coli can inactivate most of the beta-lactam antibiotics and cephalosporins and frequently demonstrate co-resistance to other antibiotics, such as aminoglycosides and quinolones [2]. The most significant factor for the development of antimicrobial resistance has been found to be selection pressure caused by antibiotics [3]. In Europe, an association between use of antimicrobial drugs and occurrence of resistance has been described at a country level [4]. Development of resistance may arise after mutations through stable genetic alterations or be an adaptive phenomenon characterised by induced tolerance when the drug is present [5]. Mechanisms of antibiotic resistance include enzymatic modification of the antibiotic, reprogramming or camouflaging the target by mutation and efflux pumps which pump the antibiotic out of the cell [6]. Carbon monoxide (CO) has been ascribed a novel role as a host defence molecule with bactericidal effects [7]. CO is produced endogenously as a result of heme metabolism through the enzyme heme oxygenase (HO) and acts as a potent regulatory and protective molecule with e.g., anti-apoptotic, anti-inflammatory and anti-proliferative effects [8]. Metal carbonyl compounds or CO-releasing molecules, CORMs, for temporal and spatial CO-delivery have been developed for therapeutic applications [9]. CO easily diffuses through membranes, while CO derived from metal carbonyl compounds may be internalized into bacteria through a Trojan horse mechanism [10], [11]. The effect of CORMs on non-pathogenic E. coli seems extensive, including actions on heme-containing proteins, and a wide range of transcriptional modifications in key metabolic pathways have been observed by CORMs [11], [12], [13], [14]. A synergistic effect of CO and the metal ion co-ligand in CORMs seems to be required for full bactericidal effect [14], [15]. Our previous results show that CORM-2 has bactericidal effects against multidrug-resistant ESBL-producing UPEC [16]. There is an urgent need for new treatment strategies suitable for targeting bacteria that are resistant to traditional antibiotics. One strategy for overcoming resistance may be to develop inhibitors of novel targets, assuming that new chemical entities are not susceptible to existing resistance mechanisms [17]. Interestingly, CORMs may be less likely to cause development of resistance mechanisms, due to multiple and different targets than existing antibiotics [9]. One of the few known carbon monoxide resistance genes is cor, which counteracts CO toxicity in Mycobacterium tuberculosis [18]. In addition, deletion of genes implicated in the process of biofilm formation (tqsA and bhsA) results in higher resistance to CORM-2 in non-pathogenic E. coli, while strains mutated in methionine related genes are hypersensitive to CORM-2 [12]. Gene profiling studies on CORMs have up to now only been carried out in non-pathogenic E. coli K12 strains [11], [12], [13], [14]. The effects of CORMs on gene expression in pathogenic bacteria, such as UPEC strains, are therefore unknown. Moreover, studies addressing the potential for bacteria to develop resistance to CORMs have not yet been performed. The aim of the present study was to use global gene profiling to assess the transcriptomic impact of CORM-2 in a multidrug-resistant ESBL-producing UPEC isolate. In addition, possible changes in gene expression, antibiotic susceptibility and virulence properties were evaluated after repeated exposure to CORM-2.


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Materials and methods Reagents CORM-2 (tricarbonyldichlororuthenium (II) dimer ([Ru(CO)3Cl2]2)) (Sigma-Aldrich, St. Louis, MO, USA) and trimethoprim (Sigma-Aldrich) were prepared by dissolution in dimethyl sulfoxide (DMSO). Cefotaxime and ciprofloxacin (Sigma-Aldrich) were prepared in sterile water. All reagents were freshly prepared or used from stock solutions.

Bacterial strains Two clinical UPEC strains, the ESBL-producing E. coli isolate 7 (ESBL7) and the non-ESBLproducing isolate UPEC2, were subjected to primary susceptibility testing through the disk dif¨ rebro University fusion method at the Department of Laboratory Medicine, Microbiology, O Hospital. ESBL7 was confirmed as ESBL-producing by detecting clavulanic acid reversible resistance for oxyiminocephalosporins and found to belong to the CTX-M-15 enzyme type and sequence type 131 [19]. ESBL7 showed resistance to cefotaxime (CTX), ceftazidime (CAZ), trimethoprim (TMP), ciprofloxacin (CIP) and mecillinam (MEL). UPEC2 was susceptible to CTX, CIP, MEL, TMP and nitrofurantoin (NIT). The commensal E. coli K12 strain MG1655 was used from laboratory stocks. The study did not involve analysis of human data, specimens or tissue samples.

Bacterial media and growth conditions Cultures were maintained on tryptic soy agar (TSA) (Becton Dickinson, Le Pont Claix, France). Overnight cultures were grown in Difco Luria-Bertani (LB) broth (Lennox; Franklin Lakes, NJ, USA) at 37˚C aerobically on a shaker at 200 rpm.

Repeated exposures to CORM-2 or vehicle Bacteria (ESBL7, UPEC2, MG1655; picked from 5–10 colonies) were suspended in 1 ml of PBS, yielding a suspension corresponding to the turbidity of McFarland 0.5, and diluted 1:100 in minimal salt (MS)-medium (~106 CFU/ml). MS-medium was prepared as previously described [20] (1.3% [wt/vol] Na2HPO4, 0.3% KH2PO4, 0.05% NaCl, and 0.1% NH4Cl supplemented with 20 mM glucose, 2 mM MgSO4, 100 μM CaCl2, and 0.25% Casamino Acids). The suspension was exposed to CORM-2 (250 μM) or vehicle (2.5% DMSO) for 4 hours at 37˚C. A volume (10 μl) was spread onto TSA-agar plates and incubated at 37˚C overnight. This procedure was repeated 10 times (10x, ~45 generations) or 20 times (20x, ~90 generations). For experimental design, see Fig 1.

RNA isolation Overnight cultures of ESBL7 from the original isolate, or isolates pre-exposed 20 times to CORM-2 or vehicle, were used to inoculate MS-medium to an optical density (OD620) of 0.1, followed by exposure to CORM-2 (250 μM) or vehicle (2.5% DMSO) for 30 min at 37˚C. RNA isolation was performed using an RNeasy mini kit (Qiagen Technologies, Hilden, Germany), according to the manufacturer’s protocol. DNA decontamination treatment was performed using Turbo DNase (Qiagen) and the quantity and purity of the purified RNA samples were determined using a spectrophotometer Nanodrop-1000 (Nanodrop Technologies Inc., Wilmington, DE, USA) by measuring the absorbance (A260, 230, 280) and calculating absorbance ratios (A260/A230 and A260/A280). All samples had A260/A230 and A260/A280 ratios above 1.9. The RNA integrity was analysed using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto,


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Fig 1. Summary of the experimental design. https://doi.org/10.1371/journal.pone.0178541.g001

CA, USA) in conjunction with RNA 6000 Nano LabChip kit (Agilent Technologies) according to the manufacturer’s protocol. RNA integrity number (RIN) values were > 8.5 for all samples.

Microarray analysis High-quality total RNA was used to prepare labelled cRNA with One-color Low Input Quick Amp WT Labelling Kit (Agilent) according to the manufacturer’s instructions. The cDNA synthesis was performed by using WT Primer Mix and cDNA Master Mix (Agilent). Labelled samples were hybridised onto G4813A E. coli gene expression Microarray 8×15K glass slides (Agilent) containing 15 208 E. coli probes. Microarrays were scanned with a G2565 CA array laser scanner (Agilent) followed by image analysis and data extraction with Feature Extraction Software version 10.7.3.1 (Agilent). Four experimental groups with 4 biological replicates in each group were analysed (total of 16 RNA samples).

Quantitative real-time PCR (qPCR) cDNA synthesis (0.1 μg of total RNA) was performed by using High Capacity cDNA Reverse Transcription Kit for single-stranded cDNA synthesis (Applied Biosystems, CA, USA) according to manufacturer‘s protocol. qPCR was performed with Maxima SYBR Green qPCR Master Mix (ThermoFisher Scientific, MA, USA) according to manufacturer’s instructions. 200–300 nM of primer and 5 ng template cDNA was added to each supermix. Primers were ordered from Eurofins MWG Synthesis GmbH (Ebersberg, Munich, Germany) (S1 Table). The RT-PCR amplification was performed in a CFX96 Touch™ Real-Time PCR Detection System (Biorad, CA, USA) using the following protocol: initial denaturation at 95˚C for 10 min, 40 cycles of denaturation at 95˚C for 15 s followed by annealing at 60˚C for 30 s and extension at 72˚C for 30 s. Each PCR was followed by a dissociation curve analysis between 60–95˚C. The Ct values were analysed by the comparative Ct (ΔΔCt) method and normalized to the endogenous control gapA (encoding glyceraldehyde 3-phosphate dehydrogenase A). Fold difference was calculated as 2-ΔΔCt.

Determination of bacterial viability after exposure to CORM-2 Overnight culture grown in LB broth was diluted 1/1000 in MS-medium (to ~106 CFU/ml) and further incubated at 37˚C on a shaker at 200 rpm to early log phase (OD620 = 0.1). The bacterial concentrations of the initial inocula used in these experiments were in the range of


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107−108 CFU/ml. Thereafter, the bacteria were exposed to CORM-2 (250–500 μM) and grown for up to 24 h in darkness at 37˚C. Time-zero samples (starting inocula) were taken and the number of viable colonies determined as described below. Samples were taken at different times after addition of CORM-2 (1, 2, 4, 8 and 24 h) depending on the experimental protocol. All samples were diluted in PBS and at least three serial dilutions were plated on TSA-plates. Following overnight culture at 37˚C, bacterial CFU/ml was determined as the mean of two dilutions. Viability was calculated as the CFU/ml in CORM-2 exposed cultures divided by the number of CFU/ml formed upon plating of the initial starting inocula and expressed as log CFU/ml.

Determination of minimum inhibitory concentration (MIC) MIC (minimum inhibitory concentration) for CORM-2, cefotaxime, ciprofloxacin and trimetophrim was determined using the broth dilution test. The test substances were inoculated with a bacterial suspension (~106 CFU/ml) in LB-broth or MS-medium (CORM-2) on 96-well plates for 18–20 h at 37˚C. All MIC tests were performed in duplicate and at least twice. The MIC was read as the lowest concentration yielding no visible growth.

Analysis of biofilm formation Overnight cultures in LB-broth were used to inoculate (at 0.1%) fresh MS-medium to an OD620 of approximately 0.05. The bacteria were seeded into 96-well plastic plates (Nunc C96 Microwell plate, Nunc A/S, Roskilde, Denmark) and exposed to CORM-2 (250 μM) or vehicle (2.5% DMSO). After 24 h of incubation under static conditions at 37˚C, biofilm formation was quantified by the crystal violet method as previously described [12]. The absorbance at 540 nm was measured by spectrophotometer (Multiscan Ascent, Thermo Labsystems, Helsingfors, Finland). The experiments were repeated three times in quadruplicate.

Motility assays Overnight cultures in LB-broth were used to inoculate (at 0.1%) fresh MS-medium to an OD620 of approximately 0.1, followed by exposure to 250 μM CORM-2 or vehicle (2.5% DMSO). Swimming motility plates (0.3% agar) and swarming motility plates (0.5% agar) were prepared as previously described [21] and bacterial suspensions were inoculated on the plates. One μl of bacterial suspension was stabbed into the swimming agar plates and 5 μl bacterial suspension spotted on swarming agar plates. The distance of migration (the diameter of the growth around the inoculation site) was measured after incubation for 14 h (swimming plates) or 20 h (swarming plates) at 37˚C. The experiments were repeated three times in duplicate.

Host renal cell activation The human renal epithelial cell line A498 (HTB-44) was obtained from American Type Culture Collection (Manassas, USA) and cultured in Dulbecco’s modified eagle medium (DMEM, Sigma-Aldrich) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM nonessential amino acids (all from Invitrogen Ltd, Paisley, UK) at 37˚C in a 5% CO2 atmosphere. During experiments, the FBS concentration was reduced to 2%. The A498 epithelial cells were stimulated with overnight cultures of ESBL7 representing the original isolate, or isolates preexposed 20 times to CORM-2 or vehicle. A multiplicity of infection (MOI) of 10 was used. Cell supernatants were collected after stimulation for 6 h and centrifuged for 5 min at 5000 x g and stored at– 80˚C. IL-6 and IL-8 cytokine production were measured using human IL-8 and IL6 kits (ELISA MAX™ Deluxe Sets, BioLegend, San Diego, CA, USA) according to


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manufacturer’s protocol and measured on a spectrophotometer (Multiscan Ascent). Cell cytotoxicity was determined using the Pierce™ LDH Cytotoxicity Assay Kit (TermoFisher Scientific, MA, USA) and absorbance measured on a spectrophotometer (Multiscan Ascent). Samples were normalized to unstimulated and lysed control cells.

Statistical analysis and microarray data processing Data are expressed as mean ± SEM. Student’s t-test was used to compare two groups and a one-way analysis of variance (ANOVA) parametric test was used for comparison of multiple groups, followed by Bonferroni multiple testing correction using the software GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). Results were considered statistically significant at p-values < 0.05. Microarray data analysis was performed using GeneSpring GX version 12.1 (Agilent) after per chip and 75th percentile shift gene normalization of samples. Statistical significant entities were obtained using the one-way ANOVA parametric test, followed by Tukey HSD post hoc test and Bonferroni FWER multiple testing correction, with a statistical significance set at a corrected p-value < 0.05 and a biological significance set at a fold change 2. Significant GO term enrichment and single experiment pathway analysis (SEA), was set at a p-value < 0.05 and < 0.1, respectively. n = number of independent experiments. Genes that exhibited a two-fold or higher increase or decrease (p < 0.05) were further classified by use of gene annotations in NCBI http://www.ncbi.nlm.nih.gov, EcoCyc http://ecocyc. org and literature mining. In addition, a virulence factor list for E. coli was generated through the PATRIC database (www.patricbrc.org), MESH virulence term association and literature mining. Gene expression data is available in the GEO database with the accession number GSE87627.

Results Analysis of transcriptional alterations in response to CORM-2 Microarray analysis was performed to analyse the gene expression alterations of ESBL7 in response to first-time exposure to CORM-2 and after pre-exposure 20 times to CORM-2. A total of 1 305 entities, common for both experimental settings, were differentially expressed with at least a two-fold change compared with vehicle (Fig 2). Of the 1 305 entities with altered transcription, 753 entities were up-regulated and 552 entities were down-regulated. Some differentially expressed gene entities were not shared between the experimental settings and were only found in response to first-time exposure to CORM-2 or after pre-exposure 20 times to CORM-2. Specific alterations in gene expression in response to first-time exposure and after exposure 20 times to CORM-2 showed 9 and 27 up-regulated and 27 and 7 down-regulated entities, respectively (Fig 2).

Gene ontology analysis Gene ontology (GO) analysis were performed on gene entities for each of the experimental settings. In total, 9 gene ontologies were enriched by the differentially expressed entities (Table 1). The enriched gene ontologies were common and found both in response to firsttime exposure to CORM-2 and after pre-exposure 20 times to CORM-2. The enriched gene ontology classes were cell communication, SOS response, cellular response to external stimulus, cellular response to extracellular stimulus, response to extracellular stimulus, fermentation, cellular response to DNA damage stimulus, DNA repair and cellular response to stress


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Fig 2. Venn diagram of differently expressed entities in ESBL isolate 7. Shown in red, first-time exposure to CORM-2 (250 μM) versus first-time exposure to vehicle (2.5% DMSO); in blue, pre-exposed 20 times to CORM-2 versus pre-exposed 20 times to vehicle. Up- and down-regulated entities are designated U and D respectively (n = 4 in each group). Overlapping regions represent entities present in both experimental conditions. https://doi.org/10.1371/journal.pone.0178541.g002

(Table 1). The differentially expressed genes enriching the different gene ontology classes are summarized in Table 2 and S2 Table.

Pathway analysis Single experiment pathway analysis (SEA) was performed in order to discover affected pathways and to further categorize the altered gene entities according to biological function. A total of 15 pathways were enriched and all were related to metabolism. Fourteen of these affected pathways were common and found both in response to first-time exposure to CORM2 and after pre-exposure 20 times to CORM-2 (Table 3). The pathway carnitine degradation I was enriched only in response to first-time exposure to CORM-2.

Alterations in gene expression common for first-time exposure to CORM-2 and pre-exposure 20 times to CORM-2 A more detailed study of the alterations in expression of virulence, antibiotic resistance and biofilm genes was performed. Some genes involved in virulence were induced following Table 1. Enriched gene ontologies, common for both first-time exposed and 20 times repeated exposure to CORM-2 (250 μM) versus vehicle (2.5% DMSO) in ESBL-producing E. coli. GO ID

GO term

Adjusted p-value

Count in selection

Count in total

No of genes up-/down-regulated

7154

cell communication

0.000

16

24

+16

9432

SOS response

0.000

16

24

+16

71496

cellular response to external stimulus

0.000

16

24

+16

31668

cellular response to extracellular stimulus

0.000

16

24

+16

9991

response to extracellular stimulus

0.000

22

41

+18/-4

6113

fermentation

0.005

17

33

+6/-11

6974

cellular response to DNA damage stimulus

0.01

20

45

+17/-3

6281

DNA repair

0.01

20

45

+17/-3

33554

cellular response to stress

0.01

22

52

+19/-3

https://doi.org/10.1371/journal.pone.0178541.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0178541 June 7, 2017

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Table 2. Differentially expressed genes of ESBL-producing E. coli following exposure to CORM-2 (250 μM) versus vehicle (2.5% DMSO). Gene

Fold change

Fold change

symbol

First exposure

20x pre-exposed

CORM-2 vs

CORM-2 vs

first exposure

20x pre-exposed

vehicle

vehicle

Gene product

Represented in all enriched ontologies polB

22.5

25.6

DNA polymerase II

sulA

13.9

15.6

suppressor of lon; inhibits cell division and ftsZ ring formation

recAa

13.6

14.2

DNA strand exchange and renaturation, DNA-dependent ATPase

yebG

12.5

15.3

DNA damage-inducible protein regulated by LexA

dinI

11.8

17.6

damage-inducible protein I

recN

11.4

13.0

protein used in recombination and DNA repair

umuD

10.2

14.5

SOS mutagenesis; error-prone repair

umuC

9.1

10.3

SOS mutagenesis and repair

lexA

8.2

10.8

regulator for SOS

ruvB

3.8

3.8

Holliday junction helicase subunit A DNA-dependent ATPase I and helicase II

uvrD

3.8

2.9

ruvA

2.5

3.3

Holliday junction helicase subunit B

uvrA

2.6

2.5

excision nuclease subunit A

uvrB

2.1

2.4

DNA repair; excision nuclease subunit B

Represented only in cell communication, SOS response, cellular response to external stimulus, cellular response to extracellular stimulus, response to extracellular stimulus, DNA repair or cellular response to stress ydjM

21.3

18.2

inner membrane protein regulated by LexA

dinB

5.2

6.1

damage-inducible protein P; putative tRNA synthetase

Represented only in cellular response to DNA damage stimulus mutM

25.2

25.8

formamidopyrimidine/5-formyluracil/ 5-hydroxymethyluracil DNA glycosylase

recF

7.5

5.7

ssDNA and dsDNA binding, ATP binding

mug

3.2

3.5

G/U mismatch-specific DNA glycosylase

phr

-3.0

-2.3

deoxyribodipyrimidine photolyase

alkB

-2.9

-2.6

DNA repair system specific for alkylated DNA

Represented only in cellular response to DNA damage stimulus ung

-2.6

-2.0

uracil-DNA-glycosylase

Represented only in response to extracellular stimulus sspB sspA

2.9

2.4

stringent starvation protein B regulator of transcription; stringent starvation protein A

2.3

2.0

yjiY

-22.9

-19.2

putative carbon starvation protein

slp

-8.2

-7.1

outer membrane protein induced after carbon starvation

psiF

-3.3

-2.3

induced by phosphate starvation

rspB

-2.9

-2.7

starvation sensing protein

Presented genes are derived from significant enrichment in the gene ontologies cell communication, SOS response, cellular response to external stimulus, cellular response to extracellular stimulus, response to extracellular stimulus, cellular response to DNA damage stimulus, DNA repair or cellular response to stress. n = 4 a

also represented in virulence

https://doi.org/10.1371/journal.pone.0178541.t002

exposure to CORM-2 (such as ibpB, recA, ycfQ), but many genes were repressed (such as kpsC, ompW, ompT, fepEG) (Table 4). Several antibiotic resistance-associated genes, such as genes coding for different multidrug efflux systems, were induced following exposure to CORM-2 (such as mdtABC, marAB, acrD) and some were repressed (such as evgA, mdtE) (Table 5).


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Table 3. Single experiment pathway analysis of ESBL-producing E. coli gene entities. Fold change Common in CORM-2 vs vehicle Pathway

p-value

Matched

Pathway

No of genes

entities

entities

up-/down-regulated

glycolysis I (from glucose-6P)

0.045

2

16

-2

glycolysis II (from fructose-6P)

0.045

2

3

-2

gluconeogenesis I

0.083

2

14

-2

glucose and xylose degradation

0.003

4

6

-3/+1

mixed acid fermentation

0.002

3

3

-1/+1a

superpathway of N-acetylneuraminate degradation

0.003

4

6

-4

superpathway of 5-aminoimidazole ribonucleotide biosynthesis

0.083

2

4

-2

superpathway of chorismate metabolism

0.079

6

24

-5/+1

superpathway of histidine, purine and pyrimidine biosynthesis

0.043

4

12

-4

superpathway of lysine degradation

0.017

2

11

-2

superpathway of phenylalanine, tyrosine and tryptophan biosynthesis

0.050

3

7

-3

superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis

0.045

2

15

-1/+1

superpathway of tryptophan biosynthesis

0.050

3

7

-3

tryptophan biosynthesis

0.002

3

9

-3

Presented pathways are affected following exposure to CORM-2 (250 μM) versus vehicle (2.5% DMSO). a part of protein complex https://doi.org/10.1371/journal.pone.0178541.t003

Some genes involved in biofilm formation, such as bhsA and yfgF encoding the anaerobic cyclic-di-GMP phosphodiesterase, were induced and some biofilm genes were repressed (Table 6). Genes involved in defence, stress response or repair, such as the gene encoding the heat shock chaperone ibpAB were markedly induced following exposure to CORM-2, while hdeA and evgA were repressed (Table 7). Three genes, hdeA, cusF (Table 7) and cusX (-58.2 first exposure; -26.3 pre-exposure 20 times) showed a significantly lower repression after preexposure 20 times to CORM-2 compared to first-time exposure. Differentially expressed genes associated with fimbriae and flagella are shown in S3 Table. CORM-2 is known to affect respiration and the majority of the differentially expressed genes involved in regulation of respiration were down-regulated (S4 Table). In order to confirm the microarray results, qPCR was carried out on five genes belonging to the functional category “antibiotic resistance” (marABR and mdtAB) and the two genes (hdeA and cusF) that showed significant differences between first-time and 20 times repeated exposure to CORM-2. In agreement with the microarray data, a marked up-regulation of marABR and mdtAB was found by qPCR (Table 8). Fold changes in mdtA expression was significantly higher after repeated exposure than after first-time exposure based on qPCR, which was not found in the microarray analysis. The microarray data showed that cusF and hdeA were significantly less repressed in cells after repeated exposure to CORM-2. qPCR data confirmed a repression of these genes but could not confirm a statistical difference between first-time and repeated exposure to CORM-2 (Table 8).

Alterations in gene expression specific for first-time exposure to CORM2 or pre-exposure 20 times to CORM-2 Although the vast majority of the differentially expressed genes were common and found both in response to first-time exposure to CORM-2 and after pre-exposure 20 times to CORM-2,


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Table 4. ESBL-producing E. coli genes associated with the functional category virulence that are differentially expressed following exposure to CORM-2 (250 μM) versus vehicle (2.5% DMSO). Gene

Fold change

Fold change

symbol

First exposure

20x pre-exposed

CORM-2 vs

CORM-2 vs

first exposure

20x pre-exposed

vehicle

vehicle

Gene product

ibpBa

2920.4

2409.3

recAb

13.6

14.2

DNA strand exchange and renaturation, DNA-dependent ATPase

ycfQ

9.9

10.6

repressor for bhsA

degP

8.2

6.2

periplasmic serine protease Do; heat shock protein HtrA

heat shock protein

oxyR

7.5

8.8

activator, hydrogen peroxide-inducible genes

flu

7.2

7.0

outer membrane fluffing protein

rdoA

6.6

6.6

Thr/Ser kinase involved in Cpx stress response

flhE

4.8

3.3

flagellar protein flhE precursor

sat

4.7

3.3

secreted auto transporter toxin

hfq

4.6

5.0

host factor I for bacteriophage Q beta replication

flhB

2.9

3.0

putative part of export apparatus for flagellar proteins

sbmA

2.6

2.5

sensitivity to microcin B17, possibly envelop protein

dsbA

2.3

3.1

fepE

-18.5

-14.6

protein disulfide isomerase I ferric enterobactin transport protein fepE

kpsC

-16.9

-21.7

KpsC protein

ompW

-16.4

-13.5

outer membrane protein W precursor

carA

-11.7

-22.3

carbamoyl-phosphate synthetase, glutamine

carB

-10.3

-10.6

carbamoyl-phosphate synthase large subunit

ompT

-10.1

-9.2

outer membrane protein 3b

chuT

-9.7

-14.1

putative periplasmic binding protein

iucA

-8.1

-7.3

IucA protein

serA

-7.7

-3.9

D-3-phosphoglycerate dehydrogenase

iucB

-7.1

-6.3

IucB protein

trpB

-6.9

-13.4

tryptophan synthase, beta protein

pyrD

-6.8

-10.4

dihydro-orotate dehydrogenase

fepG

-6.5

-11.1

ferric enterobactin transport protein

rfaL

-6.5

-5.6

O-antigen ligase

iucC

-6.3

-6.1

IucC protein

papX

-6.0

-8.5

PapX protein

flhD

-5.3

-7.5

regulator of flagellar biosynthesis

entA

-5.2

-6.0

2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase

fepC

-5.1

-5.6

ATP-binding component of ferric enterobactin transport

entB

-5.0

-6.2

2,3-dihydro-2,3-dihydroxybenzoate synthetase

evgS

-4.6

-6.4

putative sensor for regulator EvgA

entE

-4.6

-6.2

2,3-dihydroxybenzoate-AMP ligase

chuU

-4.0

-4.8

putative permease of iron compound ABC transport

chuA

-3.1

-3.7

outer membrane heme/hemoglobin receptor

csgE

-3.0

-3.3

curli production assembly/transport component

rfaP

-2.4

-2.1

lipopolysaccharide core biosynthesis

n=4 also represented in defence, stress response or repair, Table 7

a b

also represented in all enriched ontologies, Table 2



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Table 5. ESBL-producing E. coli genes associated with the functional category antibiotic resistance that are differentially expressed following exposure to CORM-2 (250 μM) versus vehicle (2.5% DMSO). Gene

Fold change

Fold change

symbol

First exposure

20x pre-exposed

CORM-2 vs

CORM-2 vs

first exposure

20x pre-exposed

vehicle

vehicle

Gene product

marA

43.9

31.4

multiple antibiotic resistance transcriptional regulator

mdtA

42.4

47.4

multidrug efflux system, subunit A

marR

37.8

33.1

multiple antibiotic resistance protein; repressor of mar operon

marB

29.5

21.0

multiple antibiotic resistance protein

mdtB

16.0

17.4

multidrug efflux system, subunit B

acrD

9.7

7.1

aminoglycoside/multidrug efflux system

ECs1864

6.7

5.1

multidrug-efflux transport protein

mdtC

5.6

8.8

multidrug efflux system, subunit C

hslJ

3.8

4.1

heat-inducible lipoprotein involved in novobiocin resistance

nfsA

3.1

3.5

nitroreductase A, modulator of drug activity A

gyrB

2.4

2.2

DNA gyrase subunit B, type II topoisomerase

rcsB

2.0

2.0

response regulator in two-component regulatory system with RcsC and YojN

evgA

-38.9

-25.0

response regulator in two-component regulatory system with EvgS

mdtE

-5.3

-4.2

anaerobic multidrug efflux transporter

tehB

-2.5

-2.8

tellurite, selenium resistance protein

a

n=4 a

also represented in defence, stress response or repair, Table 7


some specific changes were noted. Overall, the specific changes were modest with a fold change close to 2 (S5 Table). Table 6. ESBL-producing E. coli genes associated with the functional category biofilm that are differentially expressed following exposure to CORM-2 (250 μM) versus vehicle (2.5% DMSO). Gene

Fold change

Fold change

symbol

First exposure

20x pre-exposed

CORM-2 vs

CORM-2 vs

first exposure

20x pre-exposed

vehicle

vehicle

Gene product

bhsA

191.8

242.8

biofilm, cell surface and signalling protein

ydeH

93.7

100.2

diguanylate cyclase, zinc-sensing

bssS

19.7

31.3

biofilm regulator

tqsA

13.3

16.5

pheromone autoinducer 2

ybiJ

5.8

5.8

DUF1471 family putative periplasmic protein

yfgF

5.3

4.6

cyclic-di-GMP phosphodiesterase

yfaL

2.4

2.4

adhesin

artP

-5.1

-7.1

ATP-binding component of arginine transport system

bscB

-3.7

-3.8

regulator of cellulose synthase, cyclic di-GMP binding

artI

-2.9

-3.1

arginine transport system, periplasmic binding protein

csgF

-2.2

-2.9

curli production assembly/transport component

n=4 https://doi.org/10.1371/journal.pone.0178541.t006


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Table 7. ESBL-producing E. coli genes associated with defence, stress response or repair that are differentially expressed following exposure to CORM-2 (250 μM) versus vehicle (2.5% DMSO). Gene

Fold change

Fold change

symbol

First exposure

20x pre-exposed

CORM-2 vs

CORM-2 vs

first exposure

20x pre-exposed

vehicle

vehicle

Gene product

ibpBa

2920.4

2409.3

heat shock protein

ibpA

1424.7

1292.7

heat shock chaperone

spy

138.9

197.5

periplasmic ATP-independent protein refolding chaperone

frmB

86.5

48.5

S-formylglutathione hydrolase

zraP

71.5

158.7

zinc resistance protein

soxS

49.5

49.4

superoxide response regulon transcriptional activator; autoregulator

pspB

48.5

55.2

psp operon transcription co-activator

pspC

48.5

52.8

psp operon transcription co-activator

pspG

39.9

64.8

phage shock protein G

htpG

36.1

36.4

protein refolding molecular co-chaperone Hsp90, heat-shock protein

yhcN

29.9

27.2

cadmium and peroxide resistance protein

clpB

29.4

34.9

protein disaggregation chaperone

dnaK

29.2

27.2

chaperone Hsp70; DNA biosynthesis

dnaJ

28.5

21.0

chaperone with DnaK; heat shock protein

htpX

27.8

25.7

heat shock protein, integral membrane protein regulatory protein for phage-shock-protein operon

pspA

18.0

21.6

hslU

14.8

13.9

heat shock protein hslVU, ATPase subunit

norR

13.7

12.5

anaerobic nitric oxide reductase DNA-binding transcriptional activator

iscR

10.0

10.7

isc operon transcriptional repressor; suf operon transcriptional activator

grpE

8.6

11.6

heat shock protein

hslO

8.4

6.4

heat shock protein Hsp33

rpoH

8.3

10.4

RNA polymerase, sigma

loiP

7.9

9.0

Phe-Phe periplasmic metalloprotease, OM lipoprotein

iscS

7.2

4.9

putative aminotransferase

groL

6.9

7.6

GroEL, chaperone Hsp60, peptide-dependent ATPase

groS

4.6

4.4

GroES, chaperone binds to Hsp60

norW

3.6

4.3

NADH:flavorubredoxin oxidoreductase

hdeAb

-108.5

-42.6

stress response acid-resistance protein

c

evgA

-38.9

-25.0

response regulator in two-component regulatory system with EvgS

gadB

-30.9

-24.5

glutamate decarboxylase B, PLP-dependent acid resistance regulon transcriptional activator

gadX

-17.6

-16.4

cusFb

-16.6

-8.6

periplasmic copper- and silver-binding protein

gadA

-10.9

-8.0

glutamate decarboxylase A, PLP-dependent

aidB

-5.3

-4.0

DNA alkylation damage repair protein

katE

-4.2

-4.3

catalase HPII

katG

-2.2

-2.6

catalase HPI

n=4 a

also represented in virulence, Table 4

b c

significant difference between first exposure and 20x pre-exposure also represented in antibiotic resistance, Table 5



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Table 8. Quantitative real-time PCR data for ESBL-producing E. coli genes following exposure to CORM-2 (250 μM) versus vehicle (2.5% DMSO). Gene

First exposure

20x pre- exposed

symbol

CORM-2 vs

CORM-2 vs

first exposure

20x pre-exposed

vehicle

vehicle

Fold change ±

Fold change ±

SEM

SEM

Gene product

marA

26 ± 4.5

30 ± 5.9

marB

24 ± 4.1

24 ± 4.9

multiple antibiotic resistance protein

marR

40 ± 3.8

41 ± 18

multiple antibiotic resistance protein; repressor of mar operon

mdtAa

240 ± 21

370 ± 59

multidrug efflux system, subunit A

mdtB

105 ± 32

170 ± 18

multidrug efflux system, subunit B

cusF

-1.2 ± 1.4

-0.37 ± 0.84

periplasmic copper- and silver-binding protein

hdeA

-0.72 ± 1.0

0.24 ± 0.91

stress response acid-resistance protein

multiple antibiotic resistance transcriptional regulator

n=3 a

significant difference between first exposure and 20x pre-exposure


Bacterial viability in response to repeated exposure to CORM-2 Bacterial viability studies were performed to compare the growth inhibitory effect of CORM-2 (500 μM) after first-time exposure with the inhibitory effect after pre-exposure 10 or 20 times to CORM-2 or vehicle. In the viability studies, three different bacterial isolates were used: ESBL7, a non ESBL-producing UPEC isolate (UPEC2) and a commensal E. coli K12 strain (MG1655). CORM-2 (500 μM) showed a fast bactericidal effect with a reduction of bacterial counts by 4–5 log units after 1 hour of exposure in all three isolates (Figs 3A, 3C and 4A). Growth inhibition peaked after 2 hours with no resumed growth during the 24-hour study period. Untreated controls showed an increased growth response of ~2 log units during the 24-hour study period (data not shown). The inhibitory effect of CORM-2 (500 μM) did not differ significantly between samples pre-exposed to CORM-2 or samples pre-exposed to vehicle. Neither were there any significant differences in response to CORM-2 (500 μM) between bacteria exposed once, 10 or 20 times to CORM-2 (Figs 3A, 3C and 4A). A sub-MIC concentration of CORM-2 (250 μM) was examined in ESBL7 and MG1655, showing a bacteriostatic response for 4–8 hours with a recovered growth after 24 hours (Figs 3B and 4B). No significant difference in viability was found between the first-time exposure and after pre-exposure 20 times to CORM-2. To study if the recovered growth observed 24 h after exposure to 250 μM CORM-2 was caused by survival of a resistant phenotype, the bacteria were immediately re-exposed to a higher concentration of CORM-2 (500 μM). However, the sensitivity to CORM-2 (500 μM) was not reduced or dependent on the previous exposures to CORM-2 (Fig 4C).

Effect of repeated exposure to CORM-2 on cefotaxime, ciprofloxacin and trimethoprim susceptibility Determination of MIC values was performed using the broth dilution test. The MIC value for CORM-2 was determined to be 500 μM for all strains (ESBL7, UPEC2, MG1655) and MIC did not differ between first-time or repeated exposures (Table 9). Evaluation of MIC values was also performed to address whether repeated CORM-2 exposure affected the bacterial susceptibility to cefotaxime, ciprofloxacin and trimethoprim, antibiotics that are used to treat UTI. ESBL7 was resistant to cefotaxime and trimethoprim as expected, but the response to


13 / 25


Fig 3. Effects of CORM-2 exposure on viability in ESBL isolate 7 and UPEC isolate 2. A) ESBL7 and C) UPEC2 were grown to early log phase in MS-medium and then exposed to CORM-2 (500 μM) for 1, 2 or 24 h. B) ESBL7 were grown to early log phase in MS-broth and then exposed to CORM-2 (250 μM) for 1, 2, 4, 8 or 24 h. Data show viability after first-time exposure and after pre-exposure 10 or 20 times to CORM-2 (250 μM) or vehicle (2.5% DMSO). Viability is presented as log CFU/ml of CORM-2 exposed bacteria compared with the initial starting inoculum. The data are shown as mean ± SEM from three independent experiments. https://doi.org/10.1371/journal.pone.0178541.g003

ciprofloxacin was indeterminate (Table 9). The MIC values for cefotaxime, ciprofloxacin and trimethoprim in ESBL7 did not differ between the first-time exposure and after repeated exposure to CORM-2 or vehicle. The isolates UPEC2 and MG1655 were sensitive to cefotaxime, ciprofloxacin and trimethoprim. The MIC values for cefotaxime and ciprofloxacin did not change after repeated exposure to CORM-2 in UPEC2 or MG1655 (Table 9). The MIC value for trimethoprim in isolate UPEC2 remained unchanged, but strain MG1655 showed a higher MIC value (1 μg/ml vs 0.5 μg/ml) for trimethoprim after pre-exposure 20 times to CORM-2 or vehicle (Table 9).

Effect of repeated exposure to CORM-2 on biofilm formation and motility Many genes encoding biofilm were altered in response to CORM-2 and quantification of biofilm formation was performed in ESBL7 by the crystal violet method. The basal biofilm formation in ESBL7 was low (A540 ~ 0.1) and the effect of CORM-2 (250 μM) on biofilm formation was minor and not significantly different from the effect evoked by the vehicle (Fig 5A). Neither were there any significant differences in biofilm formation between bacteria exposed once or 20 times to CORM-2 (Fig 5A). Several genes encoding flagella were affected by CORM-2 and to determine whether changes in expression resulted in changed motility two motility


14 / 25


Fig 4. Effect of CORM-2 exposure on viability in E. coli K12 strain MG1655. MG1655 was grown to early log phase in MS-medium and then exposed to A) CORM-2 (500 μM) for 1, 2 or 24 h or to B) CORM-2 (250 μM) for 1, 2, 4, 8 or 24 h. C) Bacteria with a recovered growth after 24 h of exposure to 250 μM CORM-2 (see panel B) were re-exposed to a higher concentration of CORM-2 (500 μM) and the viability evaluated. Data show viability after first-time exposure and after pre-exposure 10 or 20 times to CORM-2 (250 μM) or vehicle (2.5% DMSO). Viability is presented as log CFU/ml of CORM-2 exposed bacteria compared with the initial starting inoculum. The data are shown as mean ± SEM from three independent experiments. https://doi.org/10.1371/journal.pone.0178541.g004

assays were performed. The swimming motility assay measures flagella driven individual cell movement and the swarming motility assay the flagella driven multicellular surface movement. ESBL7 developed the typical colonial patterns associated with swimming and swarming migration (data not shown). The effect of CORM-2 (250 μM) per se on motility was not significantly different from the effect evoked by the vehicle. There were no significant differences in swimming (Fig 5B) or swarming (Fig 5C) motility between bacteria exposed once or 20 times to CORM-2.

Effect of repeated exposure to CORM-2 on host renal cell production of cytokines Uroepithelial cells contribute to the initiation of host defense against UPEC through the production of various cytokines and chemokines [22]. We next addressed whether repeated CORM-2 exposure affected the ability of EBL7 to evoke IL-6 and IL-8 production from host


15 / 25


Table 9. MIC values for CORM-2, ciprofloxacin, cefotaxime and trimethoprim for ESBL-producing E. coli isolate 7, uropathogenic UPEC isolate 2 or non-pathogenic MG1655 in response to first-time exposure to CORM-2 and after 10 or 20 times pre-exposure to CORM-2 (250 μM) or vehicle (2.5% DMSO). Antibiotic susceptibility testing, MIC values First exposure CORM-2

10x CORM-2

10x vehicle

20x CORM-2

20x vehicle

CORM-2 (μM) ESBL7

500

500

500

500

500

UPEC2

500

500

500

500

500

MG1655

500

500

500

500

500

a

Breakpoint CIP (μg/ml) ESBL7

0.5

0.5

0.5

0.5

0.5

UPEC2

0.031

0.031

0.031

0.031

0.031

MG1655

0.031

0.031

0.031

0.031

0.031

>32

>32

>32

>32

>32

UPEC2

0.062

0.062

0.062

0.062

0.062

MG1655

0.062

0.062

0.062

0.062

0.062

Breakpointa 0.5/1 CTX (μg/ml) ESBL7

Breakpointa 1/2 TMP (μg/ml) ESBL7

>32

>32

>32

>32

>32

UPEC2

0.25

0.25

0.25

0.25

0.25

MG1655

0.5

0.5

0.5

1

1

Breakpointa 2/4 Abbreviations: ciprofloxacin (CIP), cefotaxime (CTX), trimethoprim (TMP) Clinical MIC breakpoint for Enterobacteriaceae set by the SRGA and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). S,

a

susceptibility/ R, resistant. -, No clinical breakpoint. https://doi.org/10.1371/journal.pone.0178541.t009

renal epithelial cells. The original ESBL7 isolate stimulated production of IL-6 and IL-8 compared to un-stimulated A498 cells (Fig 6A). The cytokine production was further increased in ESBL7 pre-exposed 20 times to CORM-2 or vehicle, but only significantly higher than the original isolate for IL-8 and CORM-2 pre-exposed bacteria (Fig 6A). The host renal cell cytotoxicity was low (~ 3%) for all experimental conditions (Fig 6B).

Discussion Gene profiling of a multidrug-resistant ESBL-producing UPEC isolate demonstrated a significant alteration of a large number of genes after exposure to the CO-donor CORM-2. In all, close to 9% of the entities on the array were altered. However, this does not correspond to a fixed number of altered genes in the genome, since multiple entities sometimes represent the same gene. Our results are in agreement with a previous transcriptome analysis of non-pathogenic E. coli where ~9% of the total genome for anaerobically grown cells and ~4% for aerobically grown cells were altered in response to CORM-2 [12]. Thus, it appears to be an extensive flux in the transcriptome of the bacteria in order to cope with the altered environment induced by CORM-2 exposure. The vast majority of the identified gene changes were common for bacteria exposed one time or repeatedly (20 times) to CORM-2. The enriched gene ontologies and pathway analysis stratified at the level common for both first-time exposed and repeatedly exposed samples


16 / 25


Fig 5. The phenotypic effect of CORM-2 on biofilm formation and motility in ESBL isolate 7. A) Biofilm formation measured after first-time exposure to CORM-2 and after pre-exposure 20 times to CORM-2 (250 μM). The biofilm formation is presented as relative changes compared to the formation evoked by the vehicle (2.5% DMSO). Motility measured on B) swimming plates and C) swarming plates after first-time exposure and after pre-exposure 20 times to CORM-2 (250 μM) or vehicle (2.5% DMSO). The motility data are presented as relative changes compared to the motility evoked by the vehicle (2.5% DMSO). Data are shown as mean ± SEM from three independent experiments. https://doi.org/10.1371/journal.pone.0178541.g005

showed that cellular responses and adaptions in metabolism genes are substantially affected. CORM-2 caused a general trend of down-regulation in energy metabolism, biosynthesis


17 / 25


Fig 6. Host renal cell production of cytokines and cytotoxicity in response to ESBL isolate 7. A) IL-6 and IL-8 production from A498 renal epithelial cells after stimulation for 6 h with the original ESBL7 isolate or with ESBL7 that have been pre-exposed 20 times to CORM-2 (250 μM) or vehicle (2.5% DMSO). B) Host renal cell cytotoxicity measured as LDH-release during the same conditions as in panel A and normalized to unstimulated and lysed control cells. Data are presented as mean ± SEM from three independent experiments. Asterisk denotes statistical significance (*p